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Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell’s cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces. 

Vascular endothelial cells (ECs) have been shown to be mechanoresponsive to the forces of blood flow, including fluid shear stress (FSS), the frictional force of blood on the vessel wall. Recent reports have shown that FSS induces epigenetic changes in chromatin. Epigenetic changes, such as methylation and acetylation of histones, not only affect gene expression but also affect chromatin condensation, which can alter nuclear stiffness. Thus, we hypothesized that changes in chromatin condensation may be an important component for how ECs adapt to FSS. Using both in vitro and in vivo models of EC adaptation to FSS, we observed an increase in histone acetylation and a decrease in histone methylation in ECs adapted to flow as compared with static. Using small molecule drugs, as well as vascular endothelial growth factor, to change chromatin condensation, we show that decreasing chromatin condensation enables cells to more quickly align to FSS, whereas increasing chromatin condensation inhibited alignment. Additionally, we show data that changes in chromatin condensation can also prevent or increase DNA damage, as measured by phosphorylation of γH2AX. Taken together, these results indicate that chromatin condensation, and potentially by extension nuclear stiffness, is an important aspect of EC adaptation to FSS. 

Ovarian cancer is routinely diagnosed long after the disease has metastasized through the fibrous submesothelium. Despite extensive research in the field linking ovarian cancer progression to increasingly poor prognosis, there are currently no validated cellular markers or hallmarks of ovarian cancer that can predict metastatic potential. To discern disease progression across a syngeneic mouse ovarian cancer progression model, here we fabricated extracellular matrix mimicking suspended fiber networks: cross-hatches of mismatch diameters for studying protrusion dynamics, aligned same diameter networks of varying interfiber spacing for studying migration, and aligned nanonets for measuring cell forces. We found that migration correlated with disease while a force-disease biphasic relationship exhibited F-actin stress fiber network dependence. However, unique to suspended fibers, coiling occurring at the tips of protrusions and not the length or breadth of protrusions displayed the strongest correlation with metastatic potential. To confirm that our findings were more broadly applicable beyond the mouse model, we repeated our studies in human ovarian cancer cell lines and found that the biophysical trends were consistent with our mouse model results. Altogether, we report complementary high throughput and high content biophysical metrics capable of identifying ovarian cancer metastatic potential on a timescale of hours. 

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a structure consisting of nesprin, SUN, and lamin proteins. A principal function of the LINC complex is anchoring the nucleus to the actin, microtubule, and intermediate filament cytoskeletons. The LINC complex is present in nearly all cell types, including endothelial cells. Endothelial cells line the innermost surfaces of blood vessels and are critical for blood vessel barrier function. In addition, endothelial cells have specialized functions, including adaptation to the mechanical forces of blood flow. Previous studies have shown that depletion of individual nesprin isoforms results in impaired endothelial cell function. To further investigate the role of the LINC complex in endothelial cells we utilized dominant negative KASH (DN-KASH), a dominant negative protein that displaces endogenous nesprins from the nuclear envelope and disrupts nuclear–cytoskeletal connections. Endothelial cells expressing DN-KASH had altered cell–cell adhesion and barrier function, as well as altered cell–matrix adhesion and focal adhesion dynamics. In addition, cells expressing DN-KASH failed to properly adapt to shear stress or cyclic stretch. DN-KASH–expressing cells exhibited impaired collective cell migration in wound healing and angiogenesis assays. Our results demonstrate the importance of an intact LINC complex in endothelial cell function and homeostasis.